Developing TB Cell Wall Enzyme Drug Targets

在本P01的项目3中，我们专注于开发和执行基于微量滴度平板的酶分析方法，用于检测结核分枝杆菌生存所需的细胞壁合成酶。在整个P01中，开发了一个精制循环，其中化合物由化合物开发模块合成，并由化合物分析模块进行分析。化合物的生物活性被报告给化合物开发模块，并重复该循环，直到产生对耐多药结核病有效且对小鼠结核病有效的先导为止。我们在项目3中的任务之一是确定化合物开发模块制造的“循环酶”抑制剂的IC50‘S，并酌情确定KI’s。目前，这些准备好循环的酶包括两种参与合成dtdp-鼠李糖的酶。 (RmlC和RmlD)和UDP-半乳糖变位酶(GIF)。由“循环酶” 目标“我们的意思是，他们已经为他们开发了分析方法，确定了晶体结构，并通过筛选化学库确定了可用作起点的抑制剂。项目3的另一项主要任务是开发四种额外的酶，以便它们也可以成为我们精炼周期的目标(即准备好循环)。这些酶是GIMU，它通过两个不同的反应合成UDP-GIcNAc，法尼基二磷酸合成酶，十一烯基二磷酸合成酶和各种半乳糖基呋喃糖基转移酶。对于这些酶，将开发基于微滴定板的分析方法，并筛选化学库以确定最初的命中率。然后，化合物开发模块将使用这种HITS与酶形成共晶，并在精炼循环的背景下设计和合成越来越活跃的抑制剂。

In project 3 of this P01, we focus on the development and execution of micro titer plate based enzyme assays for cell wall synthetic enzymes that are required by M. tuberculosis for viability. In the over all P01 a refinement cycle has been developed in which compounds are synthesized by a compound development module and analyzed by a compound analysis module. The biological activities of the compounds are reported to the compound development module and the cycle repeated until leads that are effective against MDR-TB and also effective against TB in mice are produced. One of our tasks in project 3 is to determine IC50's and, as appropriate, Ki's of inhibitors of "cycle ready enzymes" made by the compound development module. At present, these "cycle ready enzymes" include two enzymes involved in the synthesis of dTDP-rhamnose (RmlC and RmlD) and the enzyme UDP-galactopyranose mutase (GIf). By "cycle ready enzyme targets" we mean that they have already had the assays developed for them, crystal structures determined, and inhibitors that can be used as starting points identified by screening chemical libraries. Another major task of project 3 is to develop four additional enzymes so that they can also be targeted by our refinement cycle (i.e. become "cycle ready"). These enzymes are GImU which synthesizes UDP-GIcNAc via two separate reactions, farnesyl diphosphate synthetase, decaprenyl diphosphate synthetase, and various galactofuranosyl transferases. For these enzymes microtiter plate based assays will be developed and chemical libraries screened to identify initial hits. The compound development module will then use this hits to form co-crystals with the enzymes and design and synthesize increasingly more active inhibitors in the context of the refinement cycle.